The inexorable rise in the incidence of serious bacterial infections caused by multiple antibiotic-resistant bacteria in healthcare and community associated settings has become a pressing threat of public health worldwide.1 Of particular concern is the rise in incidence of methicillin resistant Staphylococcus aureus (MRSA) infections. MRSA is a human pathogen that can cause a wide range of illnesses, from mild skin and wound infections to pneumonia and bloodstream infections that cause sepsis and death. Centers for Disease Control and Prevention of United State estimates that over eighty thousand severe MRSA infections occur annually, resulting in eleven thousand deaths.2 This scenario has driven the search for novel classes of anti-staphylococcal agents which act on novel bacterial drug targets.
The bacterial cell division machinery has been considered as an important field for exploring potential novel drug targets of antibacterial agents.3 The filamenting temperature-sensitive mutant Z (FtsZ) protein undoubtedly represents one of the well-characterized and exploitable antibacterial drug targets.4 FtsZ is a cytoplasmic protein and highly conserved tubulin-like guanosine triphosphatase (GTPase), playing an important role in bacterial cell division. In order for bacteria to carry out cell division, FtsZ monomers are required to localize mid-cell through the precise positioning of cell division site positioning protein and self-polymerize into single stranded straight protofilaments by means of head-to-tail association that curve upon hydrolysis of guanosine triphosphate (GTP) molecules.5 Consecutive lateral contacts between FtsZ protofilaments produce FtsZ bundles, which eventually lead to formation of a contractile ring called Z-ring at the mid-cell. Following subsequent involvement of other downstream cell division proteins, Z-ring contraction and depolymerisation completes the cell division process to furnish identical daughter cells. Small molecules interfering initial stage of FtsZ polymerization are capable of blocking bacterial cell division, causing abrogation of bacterial cell viability eventually. These types of compounds have great potential to be developed as efficacious antimicrobial agents with a novel mode of action for clinical application. Several high resolution X-ray crystal structures of FtsZ homologs have been reported.6 These results contributed to the knowledge regarding the general organization of FtsZ protein structure, which is known to comprise two independent folding domains (FIG. 1). The N-terminal domain forms a nucleotide binding site (GTP binding site, upper red circle of FIG. 1), while the C-terminal domain contains a flexible loop (T7 loop). Both domains were interconnected via a long central helix 7 (H7) of high rigidity.
Many FtsZ-interacting compounds have been discovered and reported to bind either the GTP binding site or a cleft formed by the H7 helix, T7 loop and C-terminal β sheet (FIG. 1, lower circle). Some exhibit potent antibacterial activity with minimum inhibitory concentration (MIC) at micromolar range. PC190723 (FIG. 2)7 and its pro-drugs 1a8 and 1b9, and benzamide 210 have been demonstrated to exhibit in vitro and in vivo efficacy in a murine infection model. Moreover, X-ray crystallographic analysis revealed that PC190723 binds to a narrow cleft formed by the H7 helix, T7 loop and C-terminal β sheet (FIG. 1, lower circle).11 However, analysis of PC190723 drug resistant mutants across various MRSA strains revealed that all PC190723 drug resistant isolates had multiple mutations, resulting in amino acid substitutions that mapped to the FtsZ protein.12 These mutations mainly occurred at amino acid positions 193, 196 and 263 (FIG. 1), which accounted for over 90% of PC190723 drug resistant mutants. These results suggested that amino acid substitutions can alter slightly the overall shape of the binding pocket without interfering normal function of FtsZ. Nevertheless, this change resulted in PC190723 no longer binding to this pocket, therefore causing drug resistance. Such findings may hinder the potential of PC190723 and other related compounds13 from being developed into agents that exhibit the potential to target the same binding pocket for further clinical development. On the other hand, several compounds targeting the GTP binding site of FtsZ have also been demonstrated to exhibit potent antibacterial activity, including natural product chrysophaentin A 3,14 C8-substituted GTP analog 415, berberine analog 516 and naphthol derivative 617 (FIG. 2). Surprisingly, among these inhibitors, no drug resistant mutants have been reported in the literature so far presumably due to the fact that the GTP binding site is very important for recognizing the GTP molecule. Amino acid substitutions at this binding pocket may cause improper recognition of GTP molecule and thus hinder normal GTP hydrolysis process; therefore losing energy source to drive polymerization of FtsZ monomers.
Chan et al. identified a new class of FtsZ inhibitors exemplified by structure 7 bearing a 2,4,6-trisubstituted pyrimidine and a chiral aminoquinuclidine moiety (FIG. 2).18 Molecular docking study of compound 7 and the GTP molecule using S. aureus FtsZ revealed that the 2,4,6-trisubstituted pyrimidine moiety of 7 occupied exactly the same binding pocket as the guanosine moiety of GTP molecule through an extensive network of hydrogen bonds with FtsZ protein, suggesting that the pyrimidine moiety is crucial for binding.18 This class of compounds has been demonstrated to inhibit GTPase hydrolysis activity of S. aureus FtsZ at low micromolar IC50 value with moderate antimicrobial activity against S. aureus and E. coli. Interestingly, compound 7a also exhibited strong synergistic effect against various MRSA and vancomycin-resistant Enterococcus faecium (VREF) strains when combined with clinically used β-lactam antibiotics.19 However, structural complexity of the chiral quinuclidine scaffold and limited availability of compound have prevented further development. Accordingly, specially designed small molecules able to mimic and compete with GTP molecules to bind the GTP binding site of FtsZ may have greater potential to be developed as antimicrobial agents without acquiring drug resistance.